Spray and/or soil treatment of tobacco to reduce TSNAs

ABSTRACT

Methods for increasing the concentration of antioxidants during senescence and curing of tobacco leaves include application of a chemical solution. The methods include spraying the solution onto the tobacco plant prior to harvest and/or applying the chemical solution to the soil surrounding roots of growing tobacco plants. The application of the chemical solution preferably occurs between topping and harvest, and is optimized to stimulate the production of antioxidants and interfere with the formation of TSNAs during curing. The tobacco can be burley tobacco subjected to air curing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/443,848 filed onJan. 31, 2003, which is herein incorporated by reference in its entiretyfor all purposes.

FIELD OF INVENTION

The invention relates to methods for increasing the concentration ofadvantageous antioxidants in tobacco leaves by spraying at least onechemical solution onto a growing tobacco plant and/or soil treatment ofgrowing tobacco plants. The application of the chemical solutionpreferably occurs between topping and harvest, and can be optimized tostimulate the production of antioxidants in tobacco leaves whichinterfere with the formation of TSNAs during curing of the tobaccoleaves.

BACKGROUND OF THE INVENTION

Tobacco-specific nitrosamines (TSNAs), such as N-nitrosonornicotine(NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), can befound in smokeless tobacco; mainstream smoke; and side stream smoke ofcigarettes. It has been reported that air-cured and flue-cured tobaccocontain tobacco-specific nitrosamines. See, “Effect of Air-Curing on theChemical Composition of Tobacco”, Anna Wiernik et al., Recent Adv. Tob.Sci, (1995), 21: 39-80. According to Wiernik et al., TSNAs are notpresent in significant quantities in growing tobacco plants or fresh cuttobacco (green tobacco), but are formed during the curing process.Bacterial populations which reside on the tobacco leaves are stated tolargely cause the formation of nitrites from nitrate during curing andpossibly effect the direct catalysis of the nitrosation of secondaryamines at physiological pH values. The affected secondary amines includetobacco alkaloids, which form TSNAs when nitrosated.

Because curing of tobacco leaves is normally performed by the farmer whogrows the tobacco, a simple, economical and non-labor-intensive methodof reducing TSNA levels in the cured tobacco leaves is desirable.

SUMMARY OF THE INVENTION

According to one embodiment, a method of reducing tobacco-specificnitrosamines in cured tobacco, comprises raising the levels ofantioxidants in tobacco prior to being cured, wherein the levels ofantioxidants are raised by spraying a chemical solution onto an aerialpart (i.e., that part of the plant growing above ground) of a tobaccoplant prior to harvesting and wherein the chemical solution effects anincrease in the level of antioxidants in the tobacco plant. The chemicalsolution preferably includes a plant growth hormone such as abscicicacid (ABA) or analog thereof, a plant activator such as salicylic acid(SA) (commercially available as ACTIGARD®) or analog thereof, a plantactivator such as a harpin protein containing product commerciallyavailable as MESSENGER®, a herbicide such as methyl viologen (MV) oranalog thereof, a stress inducing agent such as hydrogen peroxide,sodium chloride (NaCl), or sulfur dioxide, or combinations thereof.Preferably, the analog of MV is Paraquat (a quaternary nitrogenherbicide widely used for weed control). Preferably, the tobacco plantsare sprayed with the chemical solution in a single application ormultiple applications about 1-3 weeks prior to harvest. Preferably, thetobacco is burley tobacco and the method further includes air curing theburley tobacco. The tobacco is preferably incorporated in a cigarette.

According to another embodiment, a method of reducing tobacco-specificnitrosamines in cured tobacco, comprises raising the levels ofantioxidants in tobacco prior to being cured, wherein the levels ofantioxidants are raised by soil treatment of tobacco plants prior toharvesting and wherein the soil treatment effects an increase in thelevel of antioxidants in the tobacco plant. The soil can be treated witha chemical solution which preferably includes a plant growth hormonesuch as abscicic acid (ABA) or analog thereof, a plant activator such assalicylic acid (SA) (commercially available as ACTIGARD®) or analogthereof, a plant activator such as a harpin protein containing productcommercially available as MESSENGER®, a herbicide such as methylviologen (MV) or analog thereof, a stress inducing agent such ashydrogen peroxide, sodium chloride, or sulfur dioxide, or combinationsthereof. Preferably, the analog of MV is Paraquat (a quaternary nitrogenherbicide widely used for weed control). Preferably, the soil is treatedin a single application or multiple applications about 1-3 weeks priorto harvest. Preferably, the tobacco is burley tobacco and the methodfurther includes air curing the burley tobacco. The tobacco ispreferably incorporated in a cigarette.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph of antioxidant capacity of green and curedOriental, bright and burley tobacco grown without a spray treatment toraise the antioxidant capacity.

FIG. 2 depicts a mechanism by which antioxidants in tobacco inhibit TSNAformation.

FIG. 3 depicts a graph of the effects on antioxidant capacity ofspraying salicylic acid and methyl viologen onto greenhouse tobacco.

DETAILED DESCRIPTION OF PREFERRED

EMBODIMENTS OF THE INVENTION

The amount of tobacco specific nitrosamines (TSNAs) in cured tobaccoleaves can be reduced by spraying growing tobacco plants with a chemicalsolution and/or treating the soil surrounding roots of growing tobaccoplants with a chemical solution. In particular, TSNAs in cured tobaccoleaves may be reduced by raising the levels of antioxidants in thetobacco leaves prior to harvesting the plants.

The antioxidant levels in tobacco leaves sprayed with the chemicalsolution are preferably raised at least 25%, preferably at least 30%,and more preferably at least 50% compared to tobacco leaves of untreatedplants grown without the spray treatment. Likewise, the antioxidantlevels in tobacco leaves of tobacco plants grown in soil treated withthe chemical solution are preferably raised at least 25%, preferably atleast 30%, and more preferably at least 50% compared to tobacco leavesof untreated plants which without the soil treatment. FIG. 1 shows anexample of the antioxidant capacity of green and cured Oriental, brightand burley tobacco obtained from untreated plants.

It is believed that TSNAs are formed predominantly during the curingprocess. While not wishing to be bound by theory, it is believed thatthe amount of TSNAs in cured tobacco leaves results from the reaction ofa reactive nitrosating species with the tobacco alkaloids.

The amount of antioxidants in tobacco leaves during the time ofair-curing is believed to be advantageous to the inhibition of TSNAformation. An elevated concentration of native antioxidants duringsenescence and air-curing of tobacco can be obtained by spraying of achemical solution preferably comprising an aqueous solution containingone or more chemical compounds onto the growing tobacco plant and/ortreating the soil surrounding roots of growing tobacco plants with theaqueous solution. For example, it is possible to increase foliarcontents of native antioxidants by stressing the tobacco plant, e.g.,using ABA to reduce the availability of carbon dioxide necessary forphotosynthesis. Preferably the tobacco is burley tobacco. The sprayingof the solution and/or soil treatment preferably occurs between topping(i.e., removal of the flower from the tobacco plant to stimulate leafproduction) and harvest, and is intended to stimulate the production ofantioxidants and interfere with the formation TSNAs during curing of thetobacco. Preferably, the level of antioxidants is increased to an amountsufficient to prevent significant nitrosation during the yellowing andbrowning phases of curing. The chemical spraying and/or soil treatmentcan be carried out only once or the tobacco plants can be sprayed and/orsubjected to soil treatment more than once. For example, the plants canbe sprayed at layby (when the plants are about knee-high) and the spraytreatment can be repeated periodically such as every 5 to 15 days untilharvest. Likewise, the soil in which the plants are grown can be treatedat layby and the soil treatment can be repeated periodically such asevery 5 to 15 days until harvest.

The spray and/or soil treatment can be combined with other methods ofelevating tobacco leaf antioxidants to a level that inhibitsnitrosation. For example, the antioxidants can be raised by pruning ofthe tobacco plants (e.g., root pruning and xylem cutting) prior toharvest. Thus, root pruning and/or xylem cutting may be used to inhibitTSNA formation during air-curing. Under the stressful conditions causedby root pruning and xylem cutting, the plant's capacity to assimilateCO₂ is reduced, and the photosynthetic electron flux to O₂ will increaseresulting in the increased production of superoxide and hydroxylradicals. These molecules induce antioxidants to cope with the oxidativestress. (See commonly-owned U.S. patent application Ser. No. 10/235,636,filed on Sep. 6, 2002, which is incorporated by reference in itsentirety herein).

Disturbances in photosynthetic activity-can be used to cause theformation of reactive oxygen species. Thus, chemicals that directlyaffect chloroplast activity can stimulate processes that induceformation of reactive oxygen species. Redox-active herbicides such asthe diphenyl ethers (e.g., acifluorfen) can act through the productionof reactive oxygen. Such herbicides can cause the activity ofantioxidants such as ascorbate, glulathione and glutathione reductase toincrease while the plant is actively defending itself against theherbicide. Bipyridyl herbicides, such as Paraquat (also known as methylviologen) and Diquat, can be used to increase oxidative stress directlyby generating reactive oxygen radicals.

Growing tobacco plants can be sprayed with a chemical solution,preferably an aqueous solution, which effects an increase in antioxidantlevels in the tobacco leaves and thus reduced TSNAs during curing of thetobacco leaves. Likewise, the soil of tobacco plants can be treated witha chemical solution, preferably an aqueous solution, which effects anincrease in antioxidant levels in the tobacco leaves and thus reducedTSNAs during curing of the tobacco leaves. Preferably, a chemicalsolution containing at least one chemical compound is sprayed ontogrowing tobacco plants using farm equipment suitable for sprayapplication of liquids to tobacco plants. If desired, spraying of suchcompounds can be combined with other nitrosamine-reducing treatmentssuch as root pruning and xylem cutting, to result in tobacco leaves withexceptionally low nitrosamine contents during the curing process.Further, the soil surrounding the roots of tobacco plants can be sprayedand/or irrigated using a chemical solution which effects the desiredincrease in the antioxidant levels in the tobacco leaves.

1. Definitions and Abbreviations

1.1 Definions

By the term “herbicide” is meant to include reagents that increaseantioxidant activity. For example, preferred herbicides include but arenot limited to redox-active herbicides and bipyridyl herbicides.Preferred redox-active herbicides include diphenyl ethers (e.g.,acifluorfen and acifluorfen sodium). Preferred bipyridyl herbicidesinclude methyl viologen (e.g., Paraquat) and Diquat.

By the term “plant growth hormone” is meant to include those plantgrowth hormones which modulate the antioxidant level and thereby TSNA inthe tobacco plant. Preferred plant growth hormones are abscicic acid(ABA) and jasmonic acid.

By the term “plant activator” is meant to include reagents whichmodulate the antioxidant level and thereby TSNA in tobacco plants. Apreferred plant activator is a salicylic acid, or an analog of salicylicacid, (e.g., ACTIGARD®), or a harpin protein (e.g., MESSENGER®).

By the term “stress inducing reagents” is meant to include reagentswhich modulate the antioxidant level and thereby TSNA in tobacco.Preferred stress inducing reagents include sulfur dioxide, sodiumchloride and hydrogen peroxide.

1.2 Abbreviations

-   -   ABA abscicic acid    -   FRAP Ferric-Reducing Antioxidant Power    -   HPLC high performance liquid chromatography    -   MV methyl viologen    -   NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone    -   NNN N-nitrosonornicotine    -   SA salicylic acid    -   TSNA tobacco-specific nitrosamines        2. Herbicides

One embodiment comprises spraying an effective amount of at least oneherbicide such as methyl viologen (MV) or one or more analogs thereof(e.g., Paraquat), or Diquat (chemical name:1′,1′-ethylene-2,2′-bipyridyldiylium) and/or other herbicides, to theaerial part of topped tobacco plants several weeks or days beforeharvesting. Also contemplated are diphenyl ethers. Preferred diphenylethers include but are not limited to acifluorfen and acifluorfensodium, which respectively have the chemical names of5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoate and sodium5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoate. Preferably,the spraying is performed one or more times in the field about 1-3 weeksbefore harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting). It hasbeen found that such spraying results in a significant reduction ofTSNAs during air-curing of burley tobacco. The reduction of TSNAs duringcuring may be accompanied by an increase in total antioxidant activity,as measured by the Ferric-Reducing Antioxidant Power assay (“FRAP”).

Another embodiment comprises applying an effective amount of at leastone herbicide such as methyl viologen (MV) or one or more analogsthereof, such as Paraquat (chemical name:1,1′-dimethyl-4,4′-bipyridinium), and/or other herbicides, to the soilsurrounding roots of topped tobacco plants several weeks or days beforeharvesting. Preferably, the soil treatment is performed one or moretimes in the field about 1-3 weeks before harvesting (i.e., 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25days before harvesting).

The herbicide(s) can be applied in a concentration of about 0.01 mM toabout 0.3 mM, more preferably 0.05 to about 0.15 mM and most preferablyin an amount of about 0.08 to about 0.11 mM (e.g., about 0.1 mMParaquat) (and every 0.01 mM unit inbetween these ranges). Typicallyabout 50 mL to about 500 mL of the solution is sprayed per plant, morepreferably an amount of about 100 mL to about 300 mL and most preferablyabout 150 mL to about 250 mL of solution is sprayed onto the leaves orthe soil of each plant.

3. Plant Activators

Another embodiment comprises spraying an effective amount of a plantactivator, such as salicylic acid (SA), or one or more analogs thereof,preferably ACTIGARD® (active ingredient chemical name:1,2,3-benzothiadiazole-7-thiocarboxylic acid-5-methyl-ester), or aharpin protein such as MESSENGER® (harpin, HrpN from Erwinia amylovora;GenBank Accession No: AAC31644), to, for example, the aerial part oftopped tobacco plants several weeks or days before harvesting. ACTIGARD®is manufactured by Syngenta Crop Protection, Greensboro, N.C. MESSENGER™is available from Eden Bioscience Corporation, Bothell, Wash.Preferably, the spraying is performed one or more times in the fieldabout 1-3 weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days beforeharvesting). For example, ACTIGARD® can be sprayed onto the tobaccoplants in a single application at a concentration of about 1 ounce (oz)per acre about 1 week prior to harvest or in a series of applications atthe same or different concentrations, e.g., 1 oz/acre, 0.5 oz/acreand/or 0.25 oz/acre. MESSENGER® can be sprayed onto the tobacco plantsin a single application at a concentration of 9 ounces per acre or in aseries of applications at the same or different concentrations.ACTIGARD® is believed to disable enzyme activity thereby creatingoxidative stress to which the tobacco plant responds by production ofantioxidants. MESSENGER® is believed to create a plant response known assystemic acquired resistance that manifests a production ofantioxidants. Due to the increase in antioxidant level in the treatedtobacco leaves, a significant reduction of TSNAs can be obtained duringcuring of the tobacco leaves.

A further embodiment comprises applying an effective amount of salicylicacid (SA), or one or more analogs thereof, preferably ACTIGARD®, or aharpin protein such as MESSENGER®, to the soil surrounding roots oftopped plants several weeks or days before harvesting. Preferably, thesoil treatment is performed one or more times in the field about 1-3weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).Salicylic acid and its analogs such as ACTIGARD® can be used to treatplants in concentrations of about 0.1 mM to about 10 mM, or morepreferably about 0.5 mM to about 2 mM and most preferably in an amountof about 0.8 mM to about 1.2 mM. Typically about 50 mL to about 500 mLof the solution is sprayed per plant, more preferably an amount of about100 mL to about 300 mL and most preferably about 150 mL to about 250 mLof solution is sprayed onto the leaves of each plant or administeredonto the soil surrounding the roots of each plant.

A harpin protein such as MESSENGER® can be used to treat the leaves orroots of the plants in an amount of about 4.5 oz/acre to about 9 oz/acre(9 oz/acre is the FDA approved amount).

4. Plant Growth Hormones

Another embodiment of the present invention comprises spraying aneffective amount of at least one plant growth hormone such as abscicicacid (ABA), or one or more of its more stable analogs, or jasmonic acidor one or more analogs thereof preferably onto the aerial part of toppedtobacco plants several weeks or days before harvesting. Preferably, thespraying is performed one or more times in the field about 1-3 weeksbefore harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting). Due tothe increase in antioxidant level in the treated tobacco leaves, asignificant reduction of TSNAs can be obtained during curing of thetobacco leaves.

ABA is a major controller of stomatal closing, and naturally increasesin concentration in tobacco leaves as a response to water-deficitstress. See FIG. 2. The CO₂ supply for photosynthesis can be interruptedleading to enhancement of the native leaf antioxidants. A solution ofABA, or a suitable analog, can be sprayed onto the tobacco plant suchthat stomata remain at least partially closed for a short time period,but then open again to support photosynthesis, akin to the action ofroot pruning, but without loss of turgor due to water deficit. Theeffects of spraying ABA on growing tobacco plants as compared to MV isshown in FIG. 3. When administering ABA onto the leaves of the plant oron the soil surrounding the roots of the tobacco plant, preferably anamount of about 0.05 mM to about 1 mM ABA is used, more preferably about0.1 to about 0.5 mM ABA and most preferably about 0.2 mM to about 0.4 mMABA.

Jasmonic acid is preferably administered to the leaves or the soilsurrounding the roots of the tobacco plants in an amount of about 0.01mM to about 2.0 mM and more preferably in an amount of about 0.1 mM toabout 1 mM.

A further embodiment of the present invention comprises applying aneffective amount of at least one plant growth hormone such as abscicicacid (ABA), or one or more of its more stable analogs or jasmonic acidor one or more analogs thereof onto the soil surrounding roots of toppedtobacco plants several weeks or days before harvesting. Preferably, thesoil treatment is performed one or more times in the field about 1-3weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).

Typically about 50 mL to about 500 mL of the solution is sprayed perplant, more preferably an amount of about 100 mL to about 300 mL andmost preferably about 150 mL to about 250 mL of solution is sprayed ontoeach plant (e.g., the leaves or the soil surrounding the roots).

5. Stress Inducing Agent

Another embodiment of the present invention comprises spraying asolution containing an effective amount of at least one stress inducingagent such as hydrogen peroxide, NaCl (salt) or sulfur dioxide onto theaerial part (the leaves) of topped tobacco plants several weeks or daysbefore harvesting. Preferably, the spraying is performed one or moretimes in the field about 1-3 weeks before harvesting (i.e., 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25days before harvesting).

A further embodiment of the present invention comprises applying asolution containing an effective amount of at least one stress inducingagent such as hydrogen peroxide, a salt (e.g., NaCl) or sulfur dioxideonto the soil surrounding the roots of topped tobacco plants severalweeks or days before harvesting. Preferably, the soil treatment isperformed one or more times in the field about 1-3 weeks beforeharvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 days before harvesting).

The foregoing methods of soil treatment and spraying one or morechemical compounds onto the leaves of the tobacco plants beforeharvesting can be combined with other methods of increasingantioxidants, to reduce nitrosamines in tobacco plants during the curingprocess. For example, partial removal of the root structure (rootpruning) of burley tobacco in the field, about a week before harvesting,results in significant reduction of tobacco-specific nitrosamines duringair-curing, probably due to the resulting increase in total antioxidantcapacity (measured by the Ferric-Reducing Antioxidant Power assay) thatpersists during the curing process.

Hydrogen peroxide treatment is preferably applied as a solutioncontaining hydrogen peroxide in a range of about 1% to about 5%, morepreferably in a range of about 2% to about 4%, and most preferably in arange of about 2.5% to about 3.5% being sprayed on the leaves and/oronto the soil surrounding the roots.

NaCl is administered to the plant by spraying on the leaves or onto thesoil surrounding the roots a solution of sodium chloride comprisingabout 0.8% to 0.1% sodium chloride, more preferably from about 0.6% toabout 0.2% NaCl, and most preferably 0.5% to about 0.3% (e.g., about0.4%) NaCl.

The sulfur dioxide is administered to the plant in an amount of about 50nL/L to about 500 nL/L, more preferably in an amount of about 100 nL/Lto about 250 nL/L and most preferably in an amount of about 150 nL/L toabout 200 nL/L.

Typically about 50 to about 500 mL of a solution with one or more ofthese reagents is sprayed per plant, more preferably an amount of about100 to about 300 mL and most preferably about 150 to about 250 mL ofsolution is sprayed onto each plant.

6. Combinations of Reagents

Also contemplated herein are combinations of reagents. For example, oneor more herbicides, stress inducing reagents, plant growth hormones, andplant activators can be combined either in an admixture or appliedseparately. The combination of reagents can be used to treat the tobaccosuch that the tobacco has increased antioxidant activity and/orproduction of antioxidants, which correspondingly reduces TSNAs.Combinations of such reagents include a plant growth hormone incombination with a plant activator (e.g., a salicylic acid combined withjasmonic acid). Additional combinations include jasmonic acid incombination with one or more stress reducing reagents, one or moreherbicides, one or more plant activators, or with a different plantgrowth hormone. When used in combinations, preferably the combinationsof reagents may require less of each reagent to be administered to thetobacco plant than if the reagent was administered alone (i.e., not incombination with other antioxidant inducing reagents).

Such combinations can be administered onto the aerial part of a tobaccoplant (topped or prior to being topped). The combination of reagents canbe administered several weeks or days before harvesting, or both.Preferably, the combination of reagents are applied by spraying. Suchspraying can be performed one or more times in the field. Preferably,the spraying of the combination of reagents occurs about 1-3 weeksbefore harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting). Thecombination of reagents can also be administered to the plant, orportions of the plant (e.g., leaves, stems), after the plant has beenharvested.

A further embodiment of the present invention comprises applying asolution containing an effective amount of a combination of reagentsonto the soil surrounding the roots of a tobacco plants (topped or priorto topping). The solution can be applied several weeks or days beforeharvesting the plant. Preferably, the soil treatment is performed one ormore times in the field about 1-3 weeks before harvesting (i.e., 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 days before harvesting). The solution can also be administered tothe plant after the plant has been harvested. Typically about 50 toabout 500 mL of the solution is sprayed per plant, more preferably anamount of about 100 to about 300 mL and most preferably about 150 toabout 250 mL of solution is sprayed onto each plant.

7. Combination of Reagents and Methods for Reducing TSNAs

The reagents discussed above can be used alone to reduce TSNAs or incombination with one another, or in combination with methods whichreduce TSNAs. Thus, the methods of treating the soil surrounding theroots or the leaves of the plants with one or more chemical compoundsbefore harvesting can be combined with other methods of increasingantioxidants, to reduce nitrosamines in tobacco plants during the curingprocess. For example, partial removal of the root structure (rootpruning) or xylem cutting of burley tobacco in the field, about a weekbefore harvesting, results in significant reduction of tobacco-specificnitrosamines during air-curing, probably due to the resulting increasein total antioxidant capacity (measured by the Ferric-ReducingAntioxidant Power assay) that persists during the curing process.

The root pruning (or other mechanical stress which produces a similarresult) induces water-deficit stress which is believed to result intemporary stomatal closure to preserve water in the plant. Such closureof the stomata also shuts off the supply of atmospheric carbon dioxidethat supports photosynthetic carbon dioxide fixation and allows theplant to produce more antioxidants. Other forms of mechanical stresswhich yield a similar result (i.e., induce production of antioxidantssuch as xylem cutting) can also be used in combination with any one orcombination of the reagents discussed above.

EXAMPLE 1 TSNA Inhibition Using Salicylic Acid or Methyl Viologen

Topped greenhouse tobacco plants were sprayed with aqueous solutions ofsalicylic acid, methyl viologen and a control substance, in order tomeasure the total antioxidant capacity (μmol/g) over 24 hours. Matureburley tobaccos (with about 20-22 leaves per plant) were topped twoweeks prior to being sprayed with either salicylic acid or methylviologen. An aqueous solution of salicylic acid (99%; Aldrich ChemicalCo.) at 1 M and methyl viologen (98%; Aldrich Chemical Co.) at 0.1 mMconcentration were sprayed upon the tobacco leaves. Each plant wassprayed with at least 200 mL of solution with good coverage of each leafto produce a more uniform response to the chemical spraying. Greensamples were taken 24 hours after spraying. Leaves were checked aftersampling to make sure all leaves are representative of the plantconditions. Samples were quickly separated into lamina and midribs asknown in the art. Only the lamina was retained for analysis. Sampledgreen lamina was flash-frozen in liquid nitrogen. The samples can bestored at −80° C. if analysis cannot be performed immediately.

Total antioxidant capacity was determined by FRAP assay, and the valueswere expressed as μmol/g of fresh weight. The concentration ofchlorogenic acid and rutin, two water soluble antioxidants, weremeasured by HPLC and-their contribution to the total antioxidantcapacity (measured by FRAP) was calculated. The results (Table 1) showthat both methyl viologen and salicylic acid increase antioxidantcapacity. TABLE 1 Antioxidant Capacity of Antioxidant Total AntioxidantChlorogenic Acid Capacity of Capacity (μmol/g) (μmol/g) Rutin (μmol/g)Control 40 12 1 Salicylic Acid 50 25 1 Methyl Viologen 55 28 1

The FRAP assay was performed as follows. The green lamina wereaccurately weighed and extracted with 10% methanol and 0.2 M perchloricacid in water. The mixture was shaken at 4° C. on an orbital shaker foran hour. The extracts were then centrifuged at 5,000 rpm for 20 minutes.The supernatant was filtered through Whatman Autovial 0.45 μm PVDFsyringe filters and was ready for analysis after appropriate dilutionwith extraction solution (10% MeOH and 0.2 M perchloric acid in water).

The procedures for the FRAP assay on tobacco samples were modified fromthat previously described by Benzie and Strain, Meth. Enzymology 299:15-27 (1999). The assay was done manually on a spectrophotometer at roomtemperature. The FRAP reagent was prepared by combining 300 mM acetatebuffer (pH 3.6), 10 mM 2,4,5-tripyridyl-s-triazine in 40 mM HCl and 20mM FeCl₃ in the ratio of 10:1:1 (v:v:v). A 100 μL aliquot of the sampleextraction was added to 3 mL of FRAP reagent and mixed. After themixture stood at room temperature for 6 min., the absorbance at 593 nmwas determined against the FRAP reagent alone. Calibration was against astandard curve (i.e., 500, 1000 and 2000 μM ferrous iron) produced bythe addition of freshly prepared ferrous sulfate. FRAP values werecalculated as micromolar ferrous ion (ferric reducing power) from twodeterminations.

Chlorogenic acid (i.e., 3-O-caffeoylquinic acid) and rutin (i.e.,quercetin 3-rhamnosidoglucoside) are two antioxidants that are watersoluble and can easily be measured in plants. Chlorogenic acid and rutinconcentrations were determined by high performance liquidchromatographic (HPLC) analysis. Other polyphenols such as the isomersof chlorogenic acid (i.e., 4-O-, and 5-O-caffeoylquinic acid) andscopoletin (i.e., 6-methoxy-7-hydroxycoumarin) were also separated byreverse phase HPLC using a gradient of methanol with 1% glacial aceticacid. 500 mg of tobacco was extracted with 10% methanol and 0.2 Mperchloric acid in water. The supernatant was neutralized using K₂CO₃ toa pH of 6.5 before the sample was injected into the HPLC. Umbelliferone(i.e. 7-hydroxycoumarin) was used as an internal standard and detectionwas by UV absorption at 326 nm.

EXAMPLE 2

The following table (Table 2) shows the results of experiments using anaqueous chemical spray to raise burley native leaf antioxidant capacityin order to interfere with TSNA production during air curing. Burleytobacco plants were grown using standard agronomic practices. ACTIGARD®and MESSENGER® were sprayed on the plants after layby in two-weekintervals with a total of five (5) applications of the reagents untilthe plants were harvested. Treatment with the ACTIGARD® or MESSENGER® atthis time in plant growth is not suitable for optimum results for themanufacturer recommended uses of those reagents as plant activators.

At harvest, care was taken to ensure clean tobacco by minimizing contactwith the soil. Plants were stalk cut and five stalks were speared oneach stick for standard curing in conventional air-curing barns. Alltreated and control tobaccos were hung with even spacing of sticks. Thetreated (non-control) tobaccos were surrounded by clean filler tobaccoto help to create normal curing conditions. At least three replicatesamples were taken at each sampling point from five different plantswith 3 leaves from the top one-third of the plant (excluding the top 4leaves). Each plant was sampled only once. Midveins were separated fromthe lamina. Both lamina and midveins were weighed and placed in samplebags stored at −80° C. After freeze-drying, samples were re-weighed todetermine the moisture content. Samples were ground to pass through a40-mesh screen and stored at −40° C. until analysis.

FRAP analysis was performed as follows. Freeze-dried samples wereaccurately weighed and extracted with 10% methanol and 0.2 M perchloricacid in water. The mixture was shaken at 4° C. on an orbital shaker foran hour. The extracts were then centrifuged at 5,000 rpm for 20 minutes.The centrifuged supernatants were then filtered through Whatman Autovial0.45 μm PVDF Syringe filters. The filtered material was then diluted asdiscussed in Example 1 above. The FRAP assay was conducted as describedby Benzie and Strain (1999) with the following modifications. The FRAPreagent was prepared daily by combining 300 mM acetate buffer (pH 3.6),10 mM 2,4,5-tripyridyl-s-triazine in 40 mM HCl and 20 mM FeCl₃ in aratio of 10:1:1 (v:v:v). A 100 μL aliquot of the sample extraction wasadded to 3,000 μL (3 mL) of FRAP reagent and mixed. After the mixturestood at room temperature for 6 minutes, the absorbance at 593 nm wasdetermined against the FRAP reagent. Calibration was against a standardcurve (i.e., 500, 1000 and 2000 μM ferrous ion) produced by the additionof freshly prepared ferrous sulfate. FRAP values were calculated asmicromolar ferrous ion (ferric reducing power) from two determinations.The total antioxidant capacity values are expressed in μmol/g of freshweight.

The treated tobaccos resulted in cured leaves with nearly 30%antioxidant capacity increases and reduction of TSNA of about 65-70% ascompared to untreated control samples. TABLE 2 ACTIGARD ® ControlMESSENGER ® 0.5 oz/ No Spray 9 oz/acre 0.25 oz/acre acre TotalAntioxidant 55.4 70.9 71.9 59.1 Capacity (μmol/g) TSNA Lamina 3040 14431787 1037 TSNA Mid-rib 5110 1057 1624 1627

EXAMPLE 3 NaCl Induced Inhibition of TSNAs

In tobacco plants, antioxidants interfere with the nitrosation ofsecondary alkaloids. The amount of antioxidants present at the time ofair-curing the tobacco plants is believed to be effective in inhibitingTSNA formation. The objective is to increase the foliar concentration ofnative antioxidants during senescence and air-curing of burley tobaccosby judicious application of reagents which inhibit TSNAs. For burleytobaccos, application preferably occurs between topping of the tobaccoplant and harvest.

High concentrations of sodium chloride (NaCl) cause ionic, osmotic andassociated secondary stresses to plants. Plant response to those primaryand secondary stresses are complex, but can be grouped into threegeneral categories: homeostasis, detoxification and growth control.

Green house grown burley tobacco plants were sprayed with either 0.7%NaCl, 0.35% NaCl, 0.18% NaCl, or control after topping one week beforeharvest. Once harvested, the leaves were examined to make sure that theleaves were representative of the plant's condition, as discussed abovefor Example 1. Samples were rapidly separated into the lamina andmidribs. The lamina containing portions were rapidly frozen in liquidnitrogen and either stored at −80° C. or analyzed. Total antioxidantcapacity was determined by FRAP assay as discussed above for Example 1and the values were expressed as μmol/g of fresh weight. TABLE 3 Control0.7% NaCl 0.35% NaCl 0.18% NaCl Total 148.4 206 229 175 antioxidantcapacity (μmol/g)

While the invention has been described with reference to preferredembodiments, it is to be understood that variations and modificationsmay be resorted to as will be apparent to those skilled in the art. Suchvariations and modifications are to be considered within the purview andscope of the invention as defined by the claims appended hereto.

1. A method of reducing tobacco-specific nitrosamines in cured tobacco,comprising raising the levels of antioxidants in tobacco leaves byspraying a chemical solution onto leaves of a growing tobacco plant atleast one time prior to harvesting, the antioxidants being raised atleast 25% compared to harvested tobacco plants grown without beingsprayed with the chemical solution.
 2. The method of claim 1, whereinthe chemical solution contains at least abscicic acid or analog thereof.3. The method of claim 1, wherein the chemical solution contains atleast salicylic acid or analog thereof.
 4. The method of claim 1,wherein the chemical solution contains at least jasmonic acid.
 5. Themethod of claim 1, wherein the chemical solution contains at leastmethyl viologen (MV) or analog thereof.
 6. The method of claim 1,wherein the chemical solution contains hydrogen peroxide, sodiumchloride or sulfur dioxide.
 7. The method of claim 1, wherein thechemical solution contains a herbicide, a plant activator, plant growthhormone and/or stress inducing agent.
 8. The method of claim 7, whereinthe herbicide, activator and/or growth hormone has an elevatedconcentration in the chemical solution compared to conventional tobaccoplant treating solutions of the same ingredients.
 9. The method of claim1, wherein the chemical solution effects reduction in available carbondioxide used in photosynthesis.
 10. The method of claim 1, furthercomprising preparing cured tobacco by harvesting the tobacco plants andsubjecting at least some of the tobacco leaves of the tobacco plants toa curing process, the level of antioxidants in the tobacco leaves beingsufficient to reduce nitrosation during the yellowing and browningphases of the curing process.
 11. The method of claim 1, wherein thechemical solution contains at least one chemical compound which canaffect chloroplast activity, the chemical compound being sprayed ontothe tobacco leaves in an amount sufficient to stimulate formation ofreactive oxygen species in the tobacco leaves.
 12. The method of claim1, wherein the chemical solution is sprayed onto the tobacco plants atdifferent times prior to harvest.
 13. The method of claim 1, wherein thetobacco is burley tobacco, the method further comprising air curing theburley tobacco after harvesting the tobacco plants.
 14. The method ofclaim 1, further comprising subjecting the growing tobacco plant tomechanical stress sufficient to raise the level of antioxidants in thetobacco leaves.
 15. The method of claim 1, wherein the tobacco plantsare sprayed only once with the chemical solution at least about one weekprior to harvesting of the tobacco plants.
 16. The method of claim 1,wherein the tobacco plants are sprayed with the chemical solutionbetween topping to remove the flowers from the tobacco plants andharvesting of the tobacco plants.
 17. The method of claim 1, furthercomprising root pruning or xylem cutting of the tobacco plants.
 18. Amethod of reducing tobacco-specific nitrosamines in cured tobacco,comprising raising the levels of antioxidants in tobacco leaves byspraying a chemical solution onto leaves of a growing tobacco plant atleast one time prior to harvesting, the antioxidants being raised atleast 25% compared to harvested tobacco plants grown without beingsprayed with the chemical solution, the chemical solution comprising anaqueous solution which effects reduction in available carbon dioxideused in photosynthesis.
 19. A method of reducing tobacco-specificnitrosamines in cured tobacco, comprising raising the levels ofantioxidants in tobacco leaves by spraying a chemical solution ontoleaves of a growing tobacco plant at least one time prior to harvesting,the antioxidants being raised at least 25% compared to harvested tobaccoplants grown without being sprayed with the chemical solution, thechemical solution comprising at least one chemical compound selectedfrom abscicic acid, salicylic acid, harpin, methyl viologen,acifluorfen, acifluorfen sodium, jasmonic acid, hydrogen peroxide,sodium chloride, and sulfur dioxide, the chemical compound being sprayedonto the tobacco leaves in an amount sufficient to stimulate formationof reactive oxygen species in the tobacco leaves.
 20. A cigarettecomprising the cured tobacco of claim
 10. 21. A method of reducingtobacco-specific nitrosamines in cured tobacco, comprising raising thelevels of antioxidants in tobacco leaves by a soil treatment wherein achemical solution is applied to the soil surrounding the roots of agrowing tobacco plant at least one time prior to harvesting, theantioxidants being raised at least 25% compared to harvested tobaccoplants grown without the soil treatment.
 22. The method of claim 21,wherein the chemical solution contains at least abscicic acid or analogthereof.
 23. The method of claim 21, wherein the chemical solutioncontains at least salicylic acid or analog thereof.
 24. The method ofclaim 21, wherein the chemical solution contains at least jasmonic acid.25. The method of claim 21, wherein the chemical solution contains atleast methyl viologen (MV) or analog thereof.
 26. The method of claim21, wherein the chemical solution contains hydrogen peroxide, sodiumchloride or sulfur dioxide.
 27. The method of claim 21, wherein thechemical solution contains a herbicide, a plant activator, plant growthhormone and/or stress inducing agent.
 28. The method of claim 27,wherein the herbicide, activator or growth hormone has an elevatedconcentration in the chemical solution compared to conventional tobaccoplant treating solutions of the same ingredients.
 29. The method ofclaim 21, wherein the chemical solution effects reduction in availablecarbon dioxide used in photosynthesis.
 30. The method of claim 21,further comprising preparing cured tobacco by harvesting the tobaccoplants and subjecting at least some of the tobacco leaves of the tobaccoplants to a curing process, the level of antioxidants in the tobaccoleaves being sufficient to reduce nitrosation during the yellowing andbrowning phases of the curing process.
 31. The method of claim 21,wherein the chemical solution contains at least one chemical compoundwhich can affect chloroplast activity, the chemical compound beingapplied to the soil in an amount sufficient to stimulate formation ofreactive oxygen species in the tobacco leaves.
 32. The method of claim21, wherein the chemical solution is applied to the soil at differenttimes prior to harvest.
 33. The method of claim 21, wherein the tobaccois burley tobacco, the method further comprising air curing the burleytobacco after harvesting the tobacco plants.
 34. The method of claim 21,further comprising subjecting the growing tobacco plant to mechanicalstress sufficient to raise the level of antioxidants in the tobaccoleaves.
 35. The method of claim 21, wherein the soil is treated onlyonce with the chemical solution at least about one week prior toharvesting of the tobacco plants.
 36. The method of claim 21, whereinthe soil is treated with the chemical solution between topping to removethe flowers from the tobacco plants and harvesting of the tobaccoplants.
 37. The method of claim 21, further comprising root pruning orxylem cutting of the tobacco plants.
 38. A method of reducingtobacco-specific nitrosamines in cured tobacco, comprising raising thelevels of antioxidants in tobacco leaves by a soil treatment wherein achemical solution is applied to the soil surrounding roots of a growingtobacco plant at least one time prior to harvesting, the antioxidantsbeing raised at least 25% compared to harvested tobacco plants grownwithout the soil treatment, the chemical solution comprising an aqueoussolution which effects reduction in available carbon dioxide used inphotosynthesis.
 39. A method of reducing tobacco-specific nitrosaminesin cured tobacco, comprising raising the levels of antioxidants intobacco leaves by a soil treatment wherein a chemical solution isapplied to the soil surrounding the roots of a growing tobacco plant atleast one time prior to harvesting, the antioxidants being raised atleast 25% compared to harvested tobacco plants grown without the soiltreatment, the chemical solution comprising at least one chemicalcompound selected from abscicic acid, saliclic acid, acifluorfen,acifluorfen sodium, harpin, methyl viologen, jasmonic acid, hydrogenperoxide, sodium chloride, and sulfur dioxide, the chemical compoundbeing applied to the soil in an amount sufficient to stimulate formationof reactive oxygen species in the tobacco leaves.
 40. A cigarettecomprising the cured tobacco of claim
 30. 41. The method of claim 4,wherein the chemical solution further comprises salicylic acid or ananalog thereof.
 42. The method of claim 4, wherein the chemical solutionfurther comprises abscicic acid, methyl viologen or an analog of methylviologen, hydrogen peroxide, sodium chloride or sulfur dioxide.
 43. Themethod of claim 4, wherein the chemical solution further comprises aherbicide, a plant growth hormone, or a stress inducing agent.
 44. Themethod of claim 4, wherein the chemical solution further comprises aherbicide, a plant growth hormone or a stress inducing agent.
 45. Themethod of claim 44, wherein the chemical solution further comprises asecond plant activator.
 46. The method of claim 4, wherein the chemicalsolution further comprises a second plant activator.
 47. The method ofclaim 1 further comprising pruning roots or cutting xylem of the tobaccoplant.
 48. The method of claim 4, further comprising pruning roots orcutting xylem of the tobacco plant.
 49. The method of claim 1, whereinthe chemical solution contains at least harpin or an analog thereof. 50.The method of claim 21, wherein the chemical solution contains at leastharpin or an analog thereof.
 51. The method of claim 1, wherein thechemical solution contains at least acifluorfen or acifluorfen sodium.52. The method of claim 21, wherein the chemical solution contains atleast acifluorfen or acifluorfen sodium.